The long term goal of this propsed research is to characterize specific changes in the structural and functional organization of cell membranes which occur with changes in hormone stimulation. In order to realize direct membrane changes, as well as specific events occurring with hormone binding, we consider it necessary to develop methods for the isolation of intact, purified plasma membranes (i.e. ghosts), or, selective membrane vesicles, from nucleated cells which represent purified plasma membranes and retain their normal enzymatic functions. Of particular interest for these hormone studies has been the retention in these membrane preparations of a hormone (catecholamine) sensitive adenylate cyclase system. The avian erythrocyte has been chosen as a model system to develop methods to isolate intact, functional plasma membranes from nucleated cells as well as purified plasma membrane vesicles. Purified membranes will be characterized for their protein composition, glycoprotein content, protein topology, enzymatic properties and their phospholipid composition. Methods to improve hormone binding studies will be pursued to further characterize the initial hormone-membrane interaction. Concomitant membrane effector events will be studied by examining membrane specific enzyme changes and using various probes such as spectroscopy to delineate subsequent membrane changes. Plasma membranes will be isolated from the human lung fibroblast, WI-38; its transformed counterpart, VA-13; and from the human astrocytoma cell line, 132-1N1, all established cell culture lines. These cell lines will be used to determine the general applicability of these methods of membrane isolation for more complex nucleated cells; to correlated general structural components from cells of various types; and, to determine both the similarities and differences in the catecholamine stimulation of these different membrane preparations. Ultimately, these studies will illuminate the relationship between membrane structural changes and membrane function.